title: An engineered HIV-1 gp41 trimeric coiled coil with increased stability and anti-HIV-1 activity: implication for developing anti-HIV microbicides.
abstract: OBJECTIVE
  We previously constructed a trimeric coiled coil, N28Fd, based on the N-heptad repeat (NHR) sequence of HIV-1 gp41, as a promising HIV-1 entry inhibitor. Here, we attempted to engineer a stabilized trimeric coiled coil, ccN28Fd, by adding interchain disulphide bonds at the N terminus of N28Fd to improve its biophysical properties and anti-HIV-1 efficacy.
  
  
  METHODS
  Molecular biology techniques were applied to engineer ccN28Fd. Circular dichroism and sedimentation velocity analysis were used to determine its secondary structure and thermostability and polymeric states, respectively. The anti-HIV-1 activity was assessed by p24 or luciferase expression. Its cytotoxicity was evaluated by XTT assay.
  
  
  RESULTS
  At low pH, ccN28Fd and N28Fd were in trimeric and monomeric conformation, respectively. ccN28Fd showed higher thermostability and much stronger antiviral activity against HIV-1 IIIB (X4) and Bal (R5) strains than N28Fd. ccN28Fd was effective in inhibiting infection by a broad spectrum of primary HIV-1 isolates and enfuvirtide-resistant strains and blocking cell-to-cell HIV-1 transmission. A combination of ccN28Fd with tenofovir, a nucleoside reverse transcriptase inhibitor-based microbicide, exhibited potent synergistic anti-HIV-1 activity. ccN28Fd was highly resistant to digestion by proteinase K at pH 7.2 and pepsin at pH 1.5, and its anti-HIV-1 activity was not significantly affected by the presence of hydroxyethyl cellulose gel, seminal fluid or vaginal fluid simulant. It possessed no significant in vitro cytotoxicity.
  
  
  CONCLUSIONS
  The engineered ccN28Fd maintains high stability in a low pH environment and exhibits potent and broad anti-HIV-1 activity, suggesting good potential for development as an effective and safe vaginal microbicide to prevent HIV sexual transmission.
authors.affiliation: School of Life Sciences, Tsinghua University, Key Laboratory for Protein Sciences of MOE, Beijing 100084, China.
authors.forename: Pei
authors.initials: P
authors.lastname: Tong
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Lu
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: Fei
authors.initials: F
authors.lastname: Yu
authors.forename: Peng
authors.initials: P
authors.lastname: Zou
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Yu
authors.forename: Yu
authors.initials: Y
authors.lastname: Li
authors.forename: Lu
authors.initials: L
authors.lastname: Lu
authors.forename: Ying-Hua
authors.initials: YH
authors.lastname: Chen
authors.forename: Shibo
authors.initials: S
authors.lastname: Jiang
mesh_terms: D000890:Anti-Infective Agents / Q000302:isolation & purification* / Q000494:pharmacology; D001688:Biological Products / Q000302:isolation & purification* / Q000494:pharmacology; D002942:Circular Dichroism; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D023581:HIV Fusion Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D015497:HIV-1; D006801:Humans; D008826:Microbial Sensitivity Tests; D015202:Protein Engineering; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D014461:Ultracentrifugation
chemical_list: D000890:Anti-Infective Agents; D001688:Biological Products; D015700:HIV Envelope Protein gp41; D023581:HIV Fusion Inhibitors; D011994:Recombinant Proteins; C514787:gp41 protein, Human immunodeficiency virus 1
keywords: N-heptad repeat; disulphide bonds; enfuvirtide; tenofovir; trimer